title: Insight into pleiotropic drug resistance ATP-binding cassette pump drug transport through mutagenesis of Cdr1p transmembrane domains.
abstract: The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Manpreet Kaur
authors.initials: MK
authors.lastname: Rawal
authors.forename: Mohammad Firoz
authors.initials: MF
authors.lastname: Khan
authors.forename: Khyati
authors.initials: K
authors.lastname: Kapoor
authors.forename: Neha
authors.initials: N
authors.lastname: Goyal
authors.forename: Sobhan
authors.initials: S
authors.lastname: Sen
authors.forename: Ajay Kumar
authors.initials: AK
authors.lastname: Saxena
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lynn
authors.forename: Joel D A
authors.initials: JD
authors.lastname: Tyndall
authors.forename: Brian C
authors.initials: BC
authors.lastname: Monk
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cannon
authors.forename: Sneha Sudha
authors.initials: SS
authors.lastname: Komath
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
mesh_terms: D019943:Amino Acid Substitution; D001693:Biological Transport, Active / Q000502:physiology; D002176:Candida albicans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025141:Drug Resistance, Fungal / Q000502:physiology*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins
keywords: ABC Transporter; ATPases; Azoles; Drug Resistance; Fungal Multidrug Resistance; Membrane Proteins; Multidrug Transporters; Pleiotropic Drug Resistance; Substrate Specificity; Transmembrane Domains